Research On The Mechanism And Experimental Therapy Of KAI1 Gene On The Inhibition Of Pancreatic Cancer Metastasis

Background and AimPancreatic cancer is one of the most common malignant tumors with insidious invasion and rapid progress. The overall 5-year survival rate of pancreatic cancer is less than 5% and metastasis has occurred in 80% patients with pancreatic cancer once the diagnosis was established. Therefore, it is very important to find an effective treatment for pancreatic cancer treatment. The etiology of pancreatic cancer is a multi-step progress resulting from the activation of some oncogenes and/or the deactivation of some cogenes. Many investigations indicate that some DNA fragments may selectively suppress the proliferation and metastasis of tumor cells in vitro. In 1995, Dong, et al. firstly cloned the gene, known as KAI1, which is relevant to the suppression of tumor metastasis from the hybridoma cell lines of prostatic carcinoma. The study of the anti-metastatic effect of KAI1 in pancreatic cancer began in 1996. Researches have shown that metastasis occurs when the expression of KAI1 gene is low or the gene is not expressed at all in pancreatic cancer. Furthermore, the mRNA level in patients with pancreatic cancer in earlier stage is remarkably higher than that with advanced pancreatic cancer. The results indicated that the gene has the ability to suppress pancreatic cancer cells mobility. Dong, et al. transfected KAI1 /CD82 gene into tumor cells to re-express CD82. The invasive and metastatic ability of tumor cells decreased after transfection. We once transfected KAI1 eukaryotic expression plasmid into pancreatic cancer cells.After transfection, the growth of pancreatic cancer cells was suppressed and the mobility decreased. We discovered that nude mice with tumor metastases in liver and lungdecreased in KAI1 teams and tumor size also diminished compared with those in control teams in vivo. Considering the disadvantages of lower transfection rate in eukaryon plasmid, we established the Ad-KAI1 adenovirus vector to carry out the experiments in vivo and in vitro. The aim of the study was to observe the suppressive effect of KAI1 on metastasis and growth of pancreatic cancer, providing further experimental evidences for the clinical treatment of pancreatic cancer.MethodsPart one: the study of the suppression of cell proliferation and metastasis of pancreatic cancer cells transfected by KAI1. (1)To establish recombination plasmid Ad-KAI1 which expressed the human tumor suppressor gene-KAI1 via adenovirus vector which lacks E1 region.(2)To compare the migration between PANC I cells teams transfected by KAI1 and control teams through Transwell experiment with the vascular endothelial growth factor(VEGF) and epidermal growth factor (EGF) inducement. (3)To detect the expression level of intercellular adhesion molecule-1 (ICAM-1) and matrix metalloproteinases-9 (MMP-9) in PANC I cells transfected by KAI1 and control teams respectively.Part two: The therapeutic research of metastatic suppression in pancreatic cancer cells in vivo. We used pancreatic cancer cell line MiaPaCa II to construct the nude mice models bearing tumors and inject the Ad-KAI1 adenovirus vector, the keno-Ad adenovirus vector and the physiological saline respectively into the animals to observe the anti- metastatic function of KAI1: (1) To detect lump volume of the three groups respectively. (2)After 50 days, to weigh their tumors, livers and lungs. (3) To compare pathomorphism of their livers, lungs and lymph nodes of the experimental group and control groups through the light microscope.ResultAn adenoviral vector carrying the full length of KAI1 (Ad-KAI1) cDNA was successfully constructed. Transwell experiment shows that there was significant difference between pancreatic cancer cells PANC I that are infected by Ad - KAI1 and control groups (P<0.05) after VEGF and EGF treatment, respectively. We found out Ad - KAI1 expressing pancreatic cancer cells significantly inhibited the migration ability of PANC I cells. Inour experiment, the growth of pancreatic cancer cells can be induced by VEGF and EGF. Positive expression of ICAM-1, MMP-9 can be detected in PANC I cells, while negative expression can be found after Ad-KAI1 infection in immunohistochemistry experiment. There is no significant difference (P>0.05) in tumor growth of pancreatic cancer bearing nude mice among Ad-KAI1 therapeutic groups, Ad groups and physiological saline groups, but KAI1 gene can inhibit the metastasis of pancreatic cancer. Considering the weight of livers and lungs, significant difference(P<0.05) showed between Ad-KAI1 groups and physiological saline groups, but no remarkable difference(P>0.05) between Ad groups and physiological saline groups. Pathological changes: We observe obvious metastasis in the surface and parenchyma of nude mice in control groups. Big volume, spherical and polygonal cells, tight arrangment , lighter cytoplasma staining, eosinphile staining and numerous caryocinesis of the cell nucleus can be seen. Metastasis forms in noduli lymphatici and bronchia. In Ad-KAI1 treatment groups, metastasis in lungs decreased with obvious smaller volume. Tumor cells are smaller and there are splits between the cells. Caryocinesis of the cell nucleus decreases and no obvious metastasis can be seen in noduli lymphatici and bronchia. Disorder arrangement of liver tumor cells with different sizes, obvious heteromporphorism and larger, irregular cell nucleus can be observed. A relative decrease in liver metastasis or none can be manifested in Ad-KAI1 treatment groups.ConclusionThe Transwell experiment and the immunohistochemistry experiment indicated that the KAI1 gene therapy can be an effective way to suppress pancreatic cancer migration if expressed in pancreatic cancer cell PANC I. The up-regulation of KAI1 expression can remarkably decrease the metastasic ability of pancreatic cancer cells. In our experiment the pancreatic cancer cell PANC I can be induced to transverse by VEGF and EGF; ICAM-1 and MMP-9 expression were inhibited in pancreatic cancer cell PANC I infected by the KAI1 gene. KAI1 gene directly injected into tumors of nude mice played an important role in inhibiting the growth and metastasis of pancreatic cancer with MiaPaca II cells. Furthermore, theresults demonstrated that KAI1 suppressed pancreatic cancer migration through inhibiting the growth of cancer cells in secondary regions.